DESCRIPTION: Ionizing radiation and other DNA damaging agents can break chromosomes. Mammalian cells are able to repair chromosomal breaks, including double-strand breaks (DSBs), although the mechanisms of the repair processes are insufficiently understood. Recent developments have led to the identification of gene products defective in ionizing radiation sensitive cell lines and to the cloning of mammalian homologs to yeast genes involved in recombinational repair of DSBs. In the past two years, this lab has demonstrated that chromosomal DSBs in mammalian cells are repaired by recombinational repair, as well as by nonhomologous mechanisms. For these studies, they have developed expression systems for a rare-cutting, site specific endonuclease in order to introduce chromosomal DSBs at repair substrates integrated into the genome. The researchers plan to exploit this system to define the pathways of recombinational repair of chromosomal DSBs in mammalian cells. They hypothesize that recombinational repair pathways will be conserved between yeast and mammalian cells. Thus, they propose to determine if recombination occurs by a conservative pathway, resulting in both crossover and noncrossover products, and if nonconservative recombination occurs, as well as if homologous crossovers occur between sister chromatids and within chromatids. They also plan to determine the effects of heterology and cell cycle stage on recombination, since two major sources of homology for repair of DSBs in mammalian genomes are divergent repetitive elements and sister chromatids. A set of cell mutants will also be screened for defects in recombinational repair. The long-term goals are to understand in detail how mammalian cells repair a major assault on their genetic integrity: the DNA DSB. Radiation can result in chromosome abnormalities and disease states. Yet, it is also a therapy in the treatments of cancers. The exchange of information between related DNA sequences (homologous recombination), apparently promoted by DSBs, is a key process leading to genetic diversity in all organisms that have been examined and plays a critical role in proper chromosome segregation at meiosis. Understanding homologous recombination and the repair of DSBs in mammalian cells, therefore, is fundamental to understanding the maintenance of integrity of the eukaryotic genome. The understanding that is gained has a long-range impact on health, medicine, and basic science.